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Abstract

Abstract The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ–hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

The gap junctions are clusters of membrane channels that allow the direct cell-to-cell passage of small cytoplasmic components, such as ions, metabolic intermediates, and second messengers, and that are thus responsible for intercellular metabolic and electrical coupling. The proteins that form these channels are encoded by a multigene family, the Cx family. The structure and assembly of Cxs into gap junctions have recently been reviewed by several authors.1234

Since the elegant experiments of Barr et al,5 it has been known that the propagation of electrical activity in the myocardium and, accordingly, the coordination of the contraction of the atrial and ventricular myocytes require the structural integrity of the gap junctions between myocytes. The physiological importance of these structures in the myocardium has given rise to numerous investigations dealing with their distribution in the different cardiac tissues, their functioning mechanism, and the regulation of their permeability.678 The identification of the Cxs that form the intermyocyte gap junctions is a first step toward a molecular understanding of the conduction phenomenon in adult and embryonic hearts.

The products of six Cx genes (Cx50, Cx46, Cx45, Cx43, Cx40, and Cx37) have been detected in the mammalian heart, and the first task that arises is to determine with which cell types, among those found in the heart (myocytes, fibroblasts, pericytes, endothelial cells, smooth muscle cells, and nerve cells), these Cxs are associated. The cardiac cells that express Cx46, a Cx whose mRNA has been detected in low amount in rat heart,9 have not yet been identified, but these cells are likely not myocytes. Cx50 was localized within the atrioventricular valves of rat heart during the postnatal period and is probably associated with interstitial cells.10 The abundance of the transcripts of the other four Cx genes (Cx45, Cx43, Cx40, and Cx37) is regulated during heart development,111213 and each of these Cxs forms homomeric gap junction channels with unique functional properties.1415 Cx43 is the major gap junction protein in the adult mammalian heart.161718 From the prenatal stages on, it has been shown to be abundantly expressed in heart, where it is associated with myocytes.11121920 However, neither Cx43 nor its mRNA has been detected in the proximal regions of the conduction system in rats,10202122 and the expression of the Cx43 gene in the nodal cells of mammalian hearts is still a matter of debate.1423 Homozygous embryos of mice lacking Cx43 survive until birth but die by asphyxiation shortly after delivery.24 Death is the consequence of an obstruction of the outflow tract, which prevents the blood flow from reaching the lungs. These findings demonstrate that, unexpectedly, Cx43 plays an essential role in cardiac morphogenesis and that at least one, or possibly several, Cxs have been able to replace Cx43 in its current-carrying capacity. Cx45 mRNA was detected in very low abundance in adult and embryonic hearts, including mouse hearts.132526 In dog hearts, Cx45 has been shown to be a component of the gap junctions of all types of myocytes (working, conductive, and nodal myocytes).2526 It was also reported to be expressed in human ventricular myocytes2728 and in cultured neonatal rat heart myocytes.29 Cx40 gene expression has been the subject of numerous studies in different mammalian species, and a feature consistently emerging from these investigations is that Cx40 is expressed in the atrial myocytes and the conductive myocytes (which form the His bundle, the two bundle branches, and the Purkinje fibers).142130 In adult mouse ventricular muscle, the distribution of Cx40 is restricted to the conduction system, and this has been demonstrated to be the consequence of the strong spatiotemporal downregulation of its expression, which occurs during development.13 A similar pattern of regulation has also been described in rat hearts.22 In addition to the atrial and conductive myocytes, Cx40 in the heart has also been detected in the endothelial cells of the coronary vessels.133132

In a previous study,13 we demonstrated that the downregulation of Cx40 gene expression, which occurs in mouse embryonic hearts from 14 dpc, correlated with the differentiation of the conduction system. Here, we have investigated the distribution of Cx40 gene products at the early stages of heart maturation, between 8.5 dpc, when the first cardiac contractions appear, and 14.5 dpc, when the formation of the four-chambered heart is almost completed. The spatiotemporal distribution of Cx40 has been compared with that of Cx43 at the same stages, and each Cx was found to be expressed with a unique pattern of distribution. We have also shown that Cx37 in heart is expressed exclusively in the endothelial cells of the endocardium and the coronary vessels, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes.

Female Swiss mice, 6 to 8 weeks of age, were mated overnight with males of the same strain. The day the vaginal plug was found was considered to be day 0.5 of pregnancy.34 The hearts were collected from 8.5- to 14.5-dpc embryos under sterile conditions. Great care was taken to ensure that only the heart was removed. The developmental stage of the embryos was checked by determining the number of somites and analyzing the morphology of the heart.35

RNA was extracted from mouse embryo hearts collected at different developmental stages using the guanidium thiocyanate method.36 Bluescript SK+ plasmids containing the complete coding regions of mouse Cx37, Cx40, or Cx433738 were propagated in Escherichia coli XL1 BLUE and purified according to standard protocols.

Preparation and Purification of Site-Directed Antibodies

The peptide (Y)GGRKSPSRPNSSASKKQ, corresponding to amino acids 315 to 331 of mouse Cx37,38 was synthesized in solid phase and conjugated to keyhole limpet hemocyanin as described previously.32 The immunization of rabbits and the affinity purification of antibodies (antibodies 315 to 331) from immune sera have been described in detail by Dupont et al.39 The preimmune sera were subjected to the same purification steps as the immune sera. The collected fractions, used in control experiments, will be referred to as preimmune fractions.

Immunoblotting Analyses

Parental HeLa cells and HeLa-Cx37 transfectants, cultured at confluence in 75-cm2 flasks, were rinsed twice with ice-cold PBS solution (pH 7.2) and harvested in 300 μL of RIPA buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing protease inhibitors (leupeptin, pepstatin, antipain, and benzamidine, at a final concentration of 1 mmol/L). The cell suspensions were frozen at −20°C, thawed, kept for 30 minutes at room temperature, and centrifuged for 10 minutes at 13 000g. The supernatants, which will be referred to as homogenates, were diluted 1:1 with the sample buffer (125 mmol/L Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol, 20 mmol/L EDTA, and 10% glycerol), and aliquots of the mixtures were fractionated by electrophoresis and electrotransferred onto nitrocellulose membranes as described previously.32 Immunoreplicas were successively incubated with rabbit antibodies 315 to 331 (10 μg/mL), biotinylated goat anti-rabbit antibodies, and peroxidase-labeled streptavidin (Jackson Immunoresearch Laboratories) as described previously.32 Peroxidase activity was revealed with H2O2 and 4-chloronaphthol according to standard procedures. The specificity of the labelings detected in the immunoreplicas was checked by (1) the analysis of nontransfected parental cell homogenates, (2) the omission of the primary antibodies (antibodies 315 to 331), (3) the replacement of the primary antibodies by a preimmune fraction, and (4) the replacement of primary antibodies by a solution of primary antibodies preincubated overnight at 4°C with the immunogenic peptide (50 μg/mL). Protein content of the cell homogenates was determined using the bicinchoninic acid assay40 (Sigma Aldrich Chimie).

Hearts collected from 14.5-dpc mouse embryos were freeze-dried and stored at −80°C until use. Samples were sonicated in a minimal volume of RIPA buffer, kept for 30 minutes at room temperature, and centrifuged for 10 minutes at 13 000g. The supernatants, referred to as homogenates, were fractionated by electrophoresis, electrotransferred as described above, and then analyzed by immunoblotting with antibodies 315 to 331 (1 μg/mL) using a chemiluminescent detection reagent kit (Boehringer-Mannheim) as previously described.13

Immunofluorescence Analyses

Antibodies

Anti-desmin mouse monoclonal antibodies (clone DE-U-10, Sigma Aldrich Chimie), diluted to 1:50, and anti-mouse CD31/PECAM-1 rat monoclonal antibodies (clone MEC 13.3; Pharmingen, Inc), diluted to 1:50, were used as markers of myocytes and endothelial cells, respectively.414243 The anti-mouse CD 31/PECAM-1 rat antibodies will be referred to hereafter as anti-PECAM antibodies. Anti-Cx43 antibodies were rabbit or hen anti-peptide antibodies, previously characterized,3244 and they were used at 3 and 8 μg/mL, respectively. The characterization of the rabbit anti-Cx40 antibodies has also been previously published.32 The rabbit anti-Cx37 antibodies were those characterized in this article (antibodies 315 to 321). The anti-Cx40 and anti-Cx37 antibodies were used at 5 and 7 to 8 μg/mL, respectively. The above primary antibodies were detected with appropriate fluorescent secondary antibodies (see below). All antibodies were diluted in a saturation solution made with PBS containing 2% (wt/vol) BSA and 0.05% (wt/vol) saponin.

Single-Labeling Experiments

Parental and Cx37-transfected HeLa cells were cultured on gelatin-coated coverslips, fixed, and permeabilized according to a previously described procedure.45 The cells were incubated overnight at 4°C with antibodies 315 to 331, washed with PBS, and incubated for 1 hour at room temperature with TRITC-labeled goat anti-rabbit IgGs (Sigma Aldrich Chimie), diluted to 1:200. The cells were carefully washed and mounted on slides with a Citifluor antifading solution (Citifluor Products). They were examined with a Zeiss III light microscope equipped for epifluorescence. The specificity of the immunolabelings in HeLa-Cx37 transfectants was checked by carrying out control experiments similar to those described above for the immunoblotting analyses.

Double-Labeling Experiments

Mouse embryos (8.5 to 9.5 dpc), fixed for 15 minutes in 2% formaldehyde solution in PBS, and isolated hearts, unfixed, collected from late embryos or adult mouse were embedded in Tissue-Tek OCT compound (Miles Laboratories) and frozen in isopentane precooled at −30°C. Frozen serial sections (5 to 10 μm thick) were mounted on gelatin-coated slides and stored at −20°C until use. The sections were incubated overnight at 4°C with rabbit anti-Cx antibodies, washed with PBS, and incubated for 1 hour at room temperature with either anti-desmin mouse monoclonal antibody or anti-PECAM rat monoclonal antibody. The sections were then washed with PBS and incubated for 1 hour with TRITC-labeled goat anti-rabbit IgGs (Sigma Aldrich Chimie) mixed with either FITC-labeled sheep anti-mouse IgGs (Sigma Aldrich Chimie) or DTAF-conjugated donkey anti-rat IgGs (Jackson Immunoresearch Laboratories). The secondary antibodies used were diluted to 1:200. The sections were processed and analyzed as described above.

Photobleach Triple-Labeling Experiments

A standard double-immunofluorescence labeling for one of the Cxs and for desmin was carried out as described above. Immunopositive cells were analyzed and imaged, and then the FITC and TRITC signals were photobleached using the light of the microscope.46 After photobleaching, a single-labeling experiment was performed using the anti-PECAM rat monoclonal antibody. The sections were then washed with PBS and incubated for 1 hour with DTAF-conjugated donkey anti-rat IgGs (Jackson Immunoresearch Laboratories). The sections were processed and analyzed as described above.

Confocal Microscopy Analysis

Frozen sections of 10.5-, 12.5-, and 14.5-dpc mouse embryonic hearts were processed for double-labeling immunofluorescence experiments with rabbit anti-Cx40 antibodies and hen anti-Cx43 antibodies as described previously.32 The primary antibodies were detected with Texas red–labeled goat anti-rabbit IgGs (Jackson Immunoresearch Laboratories) and FITC-labeled goat anti-chicken IgGs (Kirkegaard and Perry Laboratories) diluted to 1:200. The sections were analyzed by confocal laser scanning microscopy using a Leica TCS 4D instrument (Leica Lasertechnik) based on an Leitz DMRBE microscope interfaced with an argon-krypton laser adjusted to 488 and 568 nm for excitation of FITC and Texas red, respectively, and equipped with filters and detectors designed for the simultaneous recording of FITC and Texas red. The emitted signals were digitized, and pictures from screen images were printed with an NP 1600 Kodonics CS video print.

Whole-Mount In Situ Hybridization

The anti-sense and sense riboprobes were synthesized from linearized plasmids (see “Biological Material”) in the presence of UTP-digoxygenin according to the manufacturer’s instructions (Boehringer-Mannheim). The 1.06-kb mouse Cx37 anti-sense probe was synthesized with T7 polymerase from the Xba I–linearized plasmid. Mouse Cx40 and Cx43 anti-sense probes (1.35 and 0.7 kb, respectively) were both synthesized with T3 polymerase from Xho I–linearized plasmids. The Cx37 and Cx40 probes matched the complete coding regions of their respective genes3738 ; the Cx43 probe matched 400 bp of the C-terminal coding region of the mouse Cx43 gene plus 300 bp of the 3′ untranslated region.37

Whole-mount in situ hybridizations were essentially performed as described previously,47 with the following modifications: (1) The embryos were bleached with 6% H2O2 in 80% methanol for 1 hour after fixation. (2) The hybridization mix was prepared with 50% formamide, 1.3× SSC, 5 mmol/L EDTA, 0.2% Tween 20, 0.5% CHAPS, 100 μg/mL heparin, and 50 μg/mL yeast RNA. Briefly, embryos of 9.5- to 12.5-dpc stages were dissected from their decidua in PBS containing 2 mmol/L EGTA and fixed overnight at 4°C with 4% formaldehyde. Permeabilization, prehybridization, and hybridization were as described previously.47 Hybridization signals were detected by processing the embryos with alkaline phosphatase–coupled anti-digoxygenin antibodies (Boehringer-Mannheim), followed by reaction with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate. When the color was developed to the required degree, the embryos were washed three times for 5 minutes each at room temperature with TBST (140 mmol/L NaCl, 2.7 mmol/L KCl, 0.1% Tween 20, and 25 mmol/L Tris-HCl, pH 7.5), dehydrated through a methanol/TBST series, and then rehydrated by incubating the embryos through this methanol/TBST series in reverse. Before microscope analysis, the embryos were cleared for 1 hour with a 1:1 (vol/vol) glycerol/PBS containing 1 mmol/L EDTA and 0.05% Tween 20.

In some experiments, the whole-mount hybridized embryos were sectioned after color development. Stained embryos were washed for 1 hour in PBS, incubated 1 hour in 0.86% NaCl, and subjected to a series of incubations in 50%, 75%, 90%, and 100% isopropanol for 2, 3, and 6 hours and overnight, respectively. The embryos were then incubated overnight at 60°C in a mixture consisting of 1:1 (vol/vol) isopropanol/paraffin, followed by a 2-day incubation at 60°C in paraffin. Finally, the embryos were embedded in fresh paraffin, and 10-μm sections were prepared with Leica or Leitz microtomes.

Analyses of control experiments carried out with either sense probes or anti-digoxygenin antibodies did not reveal any labeling.

Identification of Cx Transcripts by RT-PCR

Primer Design and Synthesis

For the PCR and RT-PCR experiments, the primers were designed using GeneWorks software (IntelliGenetics Inc) from the murine sequences of Cx37, Cx40, Cx43, and desmin.37384849 Each pair of upstream and downstream primers had closely similar Tm values (Table 1⇓), and they were checked for minimal self-priming and upper/lower dimer formation.

Oligonucleotide Primers Used for Identification of Connexin Transcripts by RT-PCR

RT-PCR Experiments

RNA samples (2 μg per experiment), extracted from mouse embryonic heart at different developmental stages or prepared from wild-type HeLa cells, were reverse-transcripted using the murine RT (included in the First Strand cDNA Synthesis Kit, Pharmacia Biotech) with 50 pmol of each specific primer (Table 1⇑). Reactions (33 μL) were incubated for 1 hour at 37°C. Eight microliters of the first-strand reaction of each stage investigated was amplified using primer pairs specific for each of the three Cx genes or the desmin gene (Table 1⇑) in a typical 100-μL reaction containing 200 μmol/L dNTPs, 2 mmol/L MgCl2, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0 at 25°C), 0.1% Triton X-100, 100 ng of each primer, and 2.5 U Taq DNA polymerase (Promega Corp). The PCR reactions included 30 cycles with three temperature steps (94°C, 1 minute; 53°C, 1 minute; and 72°C, 1 minute) and were performed with a Perkin-Elmer-Cetus DNA thermal cycler (Roche Molecular System, Inc). One third of the amplification reaction was run in parallel with a known amount of a molecular weight marker (PM VI, Boehringer-Mannheim) on a 1.5% agarose gel stained with ethidium bromide.

All the cardiac RNA samples (2 μg) were tested for DNA contamination in reactions carried out in the absence of RT. Blank reactions were carried out without RNA in the presence of RT. RT-PCR reactions also included blank reactions in which the Taq polymerase activity was tested in the absence of cDNA preparations.

Southern Blot Analysis of Amplification Products

To check that the amplification products were those predicted, they were analyzed by Southern blot with specific radioactive probes.

DNAs, used to probe the amplicons derived from Cx37 and Cx40 mRNAs, were PCR products generated from cloned cDNAs containing the complete coding regions of mouse Cx37 and Cx40 (see “Biological Material”). The experimental conditions and the primer pairs used were similar to those described above. The amplification reactions were carried out with 100 ng of plasmid DNA. The control experiments included amplification cycles with the vectors alone, without the cloned cDNAs. The PCR products were analyzed by electrophoresis as above, and the DNA fragments were recovered from the agarose gels using the Geneclean II kit (BIO 101, Inc). These fragments of 244 bp (Cx37 probe) and 255 bp (Cx40 probe) were radiolabeled with [α-32P]dCTP by random priming using the Multiprime DNA labeling kit (Amersham International).

DNAs, used to probe the amplification products derived from Cx43 and desmin mRNAs, were synthesized oligonucleotides whose sequences, included in the amplification products, were as follows: 5′-GCCTACTCCACGGCCGGAGG-3′ (CX43 probe, positions 45 to 65; Tm, 70°C)48 and 5′-TCAACTTCCGAGAAACCAGC-3′ (desmin probe, positions 1273 to 1293; Tm, 60°C).49 These oligonucleotides were radiolabeled at their 5′ ends with [γ-32P]ATP using T4 polynucleotide kinase (Boehringer-Mannheim).

The DNA fragments amplified by RT-PCR from the RNAs, as described in the previous paragraph, were analyzed by electrophoresis in agarose gels and then transferred onto nylon membranes (Hybond N+) (Amersham International). The membranes were hybridized overnight at 65°C with the Cx37 and Cx40 probes or at 55°C with the Cx43 and desmin probes in 0.2 mol/L NaPO4 (pH 7.2), 1 mmol/L EDTA, 1% BSA, 7% SDS, and 15% formamide. The membranes were then washed at 65°C or 55°C in 40 mmol/L NaPO4 (pH 7.2), 1 mmol/L EDTA, and 1% SDS and exposed to Kodak XAR5 film (Eastman Kodak Co) for 1 hour or overnight.

Results

Detection of Cx37, Cx40, and Cx43 Transcripts by RT-PCR at the Early Stages of Heart Maturation (8.5 dpc)

At 8.5 dpc, the mouse heart, which is starting to beat,3435 is an S-shaped tube. The slow peristaltic contractions that run through it suggest that the primitive myocytes express one or more Cxs assembled into gap junction channels. In order to verify this hypothesis, the double-labeling immunofluorescence technique was applied on serial sections of 8.5-dpc embryos using antibodies specific for Cx40 or Cx43 in combination with anti-desmin or anti-PECAM antibodies, allowing identification of the myocytes or the endothelial cells, respectively.414243 Although the myocytes and the endothelial cells were strongly labeled, no immunoreactivity due to anti-Cx antibodies was observed in the heart or the vascular system. Nor were the transcripts of the three Cxs (Cx37, Cx40, and Cx43) detected in the myocardium using the in situ hybridization technique. A moderate signal was seen, however, in the paired dorsal aortas of the embryos probed for the Cx40 transcript (not shown). These results were at least partly in agreement with the previous observations of Ruangvoravat and Lo50 and Yancey et al,51 who reported having detected no Cx43 gene expression by in situ hybridization or immunofluorescence in the heart of 8.5-dpc mouse embryos. The results obtained were not altogether surprising, because the slow propagation of action potentials between embryonic myocytes52 and the synchronization of their contraction require only very few gap junction channels.535455 On the assumption that the techniques used were perhaps not sensitive enough to detect Cx gene products in the 8.5-dpc hearts, RT-PCR was applied to RNA extracted from these hearts.

RNA extracted from 8.5-, 9.5-, and 14.5-dpc embryonic hearts or from wild-type HeLa cells (in which no Cx mRNA has yet been identified)33 was reverse-transcribed using primers unique to the three Cx genes (Cx37, Cx40, and Cx43) and to the mouse desmin gene (Table 1⇑). Primer pairs unique to the above genes (Table 1⇑) were used to amplify the cDNAs generated by reverse transcription, and the resulting amplicons were analyzed by electrophoresis (Fig 1⇓). RNA samples of 14.5-dpc embryonic hearts were included in the experiments, because it had previously been demonstrated that the transcripts of Cx43, Cx40, and Cx37 were present in mouse heart at this stage of development (authors’ unpublished data, 1995). In addition, the desmin transcript was used as a positive control in the cardiac samples. Amplicons generated from 8.5-dpc embryonic heart cDNA preparations were of the predicted size (Table 1⇑) for the three Cxs and desmin (Fig 1A⇓). Amplicons of identical size were also generated from 9.5- and 14.5-dpc heart cDNAs (Fig 1B⇓ and 1C⇓). The identity of each of these amplicons was confirmed by Southern blotting (Fig 1D⇓) at the three developmental stages investigated. No DNA fragment was amplified from wild-type HeLa cell cDNA preparations (not shown). The analyses of the final reactions of the control experiments (see “Materials and Methods”) did not reveal any signal. These results indicated that Cx37, Cx40, and Cx43 gene transcripts were present in very low amount in 8.5- and 9.5-dpc hearts. The probability of the contamination of analyzed samples by noncardiac cells was extremely low, but it could not be completely ruled out.

Analyses by RT-PCR of RNA extracted from mouse embryonic heart. cDNAs synthesized by reverse transcription from RNA extracted from mouse embryonic heart of 8.5, 9.5, and 14.5 dpc (panels A, B, and C, respectively) were amplified using primer pairs unique for Cx43, Cx40, Cx37, and desmin (Table 1⇑) (lanes b, c, d, and e, respectively, in the figure). DNA standards are shown in lane a. Their sizes in base pairs were as follows: 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234/220, and 154 (this DNA fragment is barely visible). The predicted and measured sizes of the amplicons were as follows: 172, 255, 244, and 229 bp for Cx43, Cx40, Cx37, and desmin, respectively. The identity of each amplicon was confirmed by Southern blot analyses for the three developmental stages investigated. The results of these analyses for the amplicons of the 14.5-dpc embryonic stage are shown in panel D.

Cx37 Is Expressed in Both Vascular and Endocardial Endothelial Cells

From 9.5 dpc, the three Cx gene transcripts became detectable by in situ hybridization in embryos (see below). To further characterize the expression of these genes, previously characterized anti-Cx40 and anti-Cx43 antibodies were used in combination with anti-Cx37 antibodies. Characterization of the latter antibodies (also called antibodies 315 to 331) is described in the paragraph below. Characterization experiments were carried out with Cx37-transfected HeLa cells and mouse embryonic hearts. In addition to the specificity of the antibodies, these experiments demonstrated for the first time that Cx37 was expressed in the endocardial endothelial cells.

Parental HeLa cells were shown to have a very low level of gap junctional communication,56 and the above experiments demonstrated that the Cx37 gene transcript was not detectable in these cells. Analysis of parental HeLa cells by immunofluorescence and Western blotting with antibodies 315 to 331 revealed no immunoreactivity (not shown). The expression in these cells of DNA coding for murine Cx37 was shown to induce high electrical and dye coupling, thus providing evidence of the formation of functional gap junction channels upon transfection.33 In the immunoreplicas of HeLa-Cx37 transfectant homogenates, probed with antibodies 315 to 331, Cx37 was detected as a single-band protein with an Mr of 37 000 (Fig 2A⇓, lane b). This was similar to the Mr deduced from immunoprecipitation experiments carried out with in vitro translation products of rat Cx37 mRNA.57 No labeling was detected in immunoreplicas probed with either preimmune fractions (Fig 2A⇓, lane c) or antibodies 315 to 331 preincubated with the immunogenic peptide (Fig 2A⇓, lane d). Analysis of HeLa-Cx37 transfectants by fluorescence microscopy showed that immunoreactivity due to antibodies 315 to 331 is localized both in the intercellular contact sites, where the gap junctions usually occur, and in cytoplasmic vesicles, which accumulate at one pole of the nucleus (Fig 2B⇓ and 2C⇓). Recent investigations have shown that the overexpression of Cxs induces the aberrant formation of gap junctions in various cell compartments, including the nuclear envelope and the endoplasmic reticulum.58 Thus, the presence of immunoreactive sites in cytoplasmic vesicles of transfected HeLa cells probably reflects the overexpression of the Cx.

Characterization of rabbit antibodies raised to residues 315 to 331 of mouse Cx37 (antibodies 315 to 331 or anti-Cx37 antibodies). A, Western blots. Protein (100 μg) was loaded in the wells (lanes b through f) before electrophoresis and electrotransfer. Lane a shows mobility of molecular mass standards, which are indicated in kilodaltons (phosphorylase a, Mr of 94 000; BSA, Mr of 67 000; ovalbumin, Mr of 43 000; carbonic anhydrase, Mr of 29 000; and soybean trypsin inhibitor, Mr of 21 000 [lysosyme, Mr of 14 000, is not shown]). Lanes b, c, and d show replicas of HeLa-Cx37 transfectant homogenates probed with antibodies 315 to 331. Cx37 was detected as a single protein with Mr of 37 000 (lane b). No protein was detected in replicas incubated with either antibodies 315 to 331 preincubated with the immunogenic peptide (lane c) or preimmune fractions (lane d). Lane e shows a replica of a homogenate prepared from 14.5-dpc mouse embryonic heart and probed with antibodies 315 to 321. A single protein that migrated with the same mobility as Cx37 in transfected HeLa cells was detected. Lane f shows a replica of homogenate prepared from 14.5-dpc mouse embryonic heart and probed with a preimmune fraction. B and C, Characterization by immunofluorescence of antibodies 315 to 321 in HeLa-Cx37 transfectants. Panel B shows a phase-contrast micrograph of HeLa-Cx37 transfectants (bar=15 μm). Nucleoli appear as dark spots in the nuclei. Small arrows indicate intercellular domains that are labeled with antibodies 315 to 321 as shown in panel C. Panel C shows fluorescence microscopy of the cells shown in panel B. In some cells, Cx37 is observed in cytoplasmic vesicles gathered at one pole of the nuclei. Small arrows indicate intercellular contacts that are labeled with antibodies 315 to 321. No labeling was detected in transfectants processed according to the control experiment protocols. Panels D, E, and F show double-labeling immunofluorescence experiments with antibodies 315 to 331 and anti-PECAM antibodies from a frozen section in a 14.5-dpc mouse embryo. The pulmonary trunk is seen in the center of panel D (phase-contrast micrograph, bar=25 μm), surrounded by mesenchymal cells. The black star in panel D indicates the lumen of the vessel, which contains a few blood cells (asterisks). The endothelial cells that delimit the lumen are clearly identified by their immunoreactivity to anti-PECAM antibody (panel E). In the same section, antibodies 315 to 331 reveal small immunoreactive sites (panel F), which are associated with endothelial cells (small arrows in panels D, E, and F).

In the mouse heart, the abundance of Cx37 mRNA is much higher during fetal life than at the adult stage,13 and accordingly, the protein should be more abundant at the embryonic than at the adult stages. Thus, the characterization of antibodies 315 to 331 was continued using mouse embryonic heart. In the immunoreplicas of 14.5-dpc embryonic heart homogenates probed with antibodies 315 to 331, a single protein was detected (Fig 2A⇑, lane e). Its electrophoretic mobility matched that of Cx37 expressed in HeLa transfectants (Fig 2A⇑, lane b). No protein was revealed in replicas incubated with either preimmune fractions (Fig 2A⇑, lane f) or antibodies preincubated with the immunogenic peptide (not shown). Double-labeling immunofluorescence experiments, carried out with 14.5-dpc embryonic heart sections, using antibodies 315 to 331 and anti-PECAM antibody, showed that immunoreactivity due to antibodies 315 to 331 was exclusively associated with intercellular domains of the endothelial cells of both the blood vessels and the endocardium (Fig 2D⇑ through 2F; also see Fig 10C⇓). Specific labelings of vascular and endocardial endothelial cells were also observed in younger embryos (9.5 dpc, vascular endothelial cells, Fig 4A⇓ and 4D⇓; 10.5 dpc, endocardial endothelial cells, Fig 8D⇓ through 8F; and 13.5 dpc, endocardial endothelial cells, Fig 6G⇓ through 6H). No immunofluorescence signal was ever observed in embryo sections or in Cx37-transfected HeLa cells using preimmune fractions or antibodies 315 to 331 preincubated with the immunogenic peptide (not shown).

The results described above demonstrate that antibodies 315 to 331 are specific for Cx37, and they will hereafter be referred to as anti-Cx37 antibodies. They were used in the work described below to investigate the distribution of Cx37 in the cardiovascular system.

As reported above, the Cx gene products became detectable by in situ hybridization in the embryos from 9.5 dpc. From this stage until the 12.5-dpc stage, distribution of transcripts was investigated by in situ hybridization. Protein expression was studied until 14.5 dpc by double- and triple-labeling immunofluorescence techniques using anti-Cx antibodies (anti-Cx37, -Cx40, and -Cx43) in combination with anti-desmin and anti-PECAM antibodies. For the sake of clarity, gene expression will be described first in the vascular system (next paragraph) and then in the heart, where atrial and ventricular tissues will be examined separately.

Cx40 and Cx37 Are Coexpressed in the Vascular Endothelial Cells From 9.5 dpc

At 9.5 dpc, the most patent result of in situ hybridization experiments was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes (Fig 3A⇓ and 3B⇓). By contrast, Cx43 transcript was never detected in vessels whatever the developmental stage investigated (Fig 3C⇓). Cx40 transcript was highly expressed in the vasculature and was observed in the paired dorsal aortas, the intersegmental arteries sprouting from the aortas, the brachial arch arteries, the primary head arteries and branching vessels, the atrium, and the common ventricular chamber (Fig 3A⇓, 3D⇓, 3E⇓, and 3F⇓). More caudally, Cx40 transcript was detected in the common aorta and the vitelline, umbilical, and caudal vessels (Fig 3A⇓ and 3E⇓). During development, between 9.5 and 12.5 dpc, the vascular expression pattern of Cx40 transcript was modified after the rearrangement of the vasculature. Analyses of sections prepared from 9.5- to 12.5-dpc embryos hybridized with anti-sense Cx40 riboprobe (Fig 3G⇓ and 3I⇓) and sections incubated with anti-Cx40 antibodies (Fig 4C⇓ and 4F⇓) demonstrated that Cx40 gene products were expressed in the endothelial cells of the vessels. At 9.5 dpc, Cx40 transcript was also seen to be expressed, with a periodic pattern, in the lateral myoblasts of the anterior region of embryos (Fig 3A⇓), as previously described by Dahl et al59 in the mouse and Van Kempen et al22 in the rat, at later developmental stages. The presence of this transcript in myoblasts was observed until the last stage analyzed by in situ hybridization, ie, 12.5 dpc (E. Dahl and K. Willecke, unpublished data, 1997).

Whole-mount in situ hybridization. A, B, and C, Left lateral views of 9.5-dpc mouse embryos hybridized with probes specific for Cx40, Cx37, and Cx43 gene transcripts, respectively. Panel A shows that Cx40 transcript is highly expressed in the paired dorsal aortas (only the left branch is visible), the intersegmental arteries sprouting from the aortas, the branchial arch arteries (I, II, III, and IV), the primary head arteries and branching vessels, and the atrium (a). In some embryos, a weak hybridization signal was sometimes detectable in the common ventricular chamber (v) (see panel B in Fig 5⇓). Caudally, the transcript is detected in the vitelline vessels at the posterior end of the common aorta and in the umbilical (u) and caudal vessels. Weaker signals are also seen in the myoblasts (m). Panel B shows that Cx37 transcript is detected in the vascular system as early as 9.5 dpc, with a distribution similar to that of the Cx40 transcript. In heart, the hybridization signal is seen in both the atrium (a) and the ventricular chamber (left of the atrium). Visualization of the Cx37 transcript always required a color development time longer than for the Cx40 transcript, suggesting that the Cx37 transcript was less abundant than the Cx40 transcript. Panel C shows the distribution of the Cx43 gene transcript in a whole-mount hybridized embryo. No hybridization signal is seen in the heart (h) or the vascular system. Hybridization signals are found in the nasal placodes (np), the diencephalon (d), the optic vesicles (ov), the metencephalon (m), the auditory vesicles (av), the mandibular arch (ma), the dorsal somites (s), the anlage of the forelimbs (fl), and the caudal neural folds (f), in agreement with previous descriptions.5051 D, E, and F, Details of Cx40 gene transcript expression in the vasculature of 9.5-dpc embryos. In panel D, the transcript is clearly seen in the left and right branches of the dorsal aorta, the head vessels, the arteries of branchial arches (I, II, and III), and the atrium (bottom of the right corner). In panel E, details of the small intersegmental arteries and of the fusion of the branches of the paired dorsal aortas into the common aorta are seen. In panel F, the left lateral view of the intersegmental arteries in the dorsal midregion of an embryo is shown. Periodic weak intensity signals correspond to the right intersegmental arteries seen by transparency through the embryo. G and I, Transverse sections in a whole-mount hybridized 10.5-dpc embryo. Note in panel G the labeling of the paired dorsal aortas (ao) and intersegmental arteries (ia). Panel I shows, in another section at higher magnification, the labeling of endothelial cells in the right branch of the paired dorsal aortas. Bars=50 and 25 μm in panels G and I, respectively. H and J, Transverse sections in a whole-mount hybridized 10.5-dpc embryo. Note in panel H the labeling of the common aorta. Panel J shows, in another section at higher magnification, the labeling of E cells in the left branch of the paired dorsal aortas. Bars=100 and 25 μm in panels H and J, respectively.

Expression of Cx40 and Cx37 in vascular endothelial cells from frozen sections in 9.5-dpc embryos. A through C, Transverse sections at the level of the paired dorsal aortas. The section shown in panels A and B was treated with both anti-Cx37 and anti-PECAM antibodies (double-labeling immunofluorescence experiments). Note the labeling of endothelial cells of aortas (arrows) with both antibodies. A similar labeling of aortas is observed in an adjacent section (panel C) incubated with anti-Cx40 antibodies. Collapse of the paired dorsal aortas is due to the freezing process. nt indicates neural tube. Bar in panel A=40 μm (representative of panels A through C). D through F, Transverse sections in the anterior part of an embryo through the maxillary component of the first branchial arch (arrows in panel D). Asterisks indicate cross sections of the first branchial arch arteries. The section shown in panels E and F was treated with both anti-PECAM and anti-Cx40 antibodies (double-labeling immunofluorescence experiment). Note the labeling of endothelial cells in arteries. A similar labeling is observed in an adjacent section (panel D) incubated with anti-Cx37 antibodies. Bar in panel D=40 μm (representative of panels D through F).

Cx37 transcript was detected both in the vessels and in the heart, but hybridization signals were weaker than for Cx40 transcript (Fig 3B⇑). Its distribution in the vascular system was similar to that described above for Cx40 transcript. Analyses of vessels present in sections prepared from embryos hybridized with Cx37 anti-sense riboprobe indicated that the transcript was expressed in endothelial cells (Fig 3H⇑ through 3J). Expression of the protein in the same cells was demonstrated by immunofluorescence experiments (Fig 4A⇑ and 4D⇑). Cx37 was never observed in myocytes whatever the stage investigated, including the adult stage.

Cx40 Gene Is Activated Earlier Than Cx43 Gene in Atrial Myocytes

At 9 dpc, the looping process of the primitive heart is completed, placing the cardiac chambers near their final relative position. By 9.5 dpc, the primitive atrium is a single chamber not yet divided into right and left parts but separated from the common ventricular chamber by the constriction of the atrioventricular canal. At this stage, in situ hybridization experiments showed that Cx40 transcript was clearly detectable in the thin atrial wall formed by two or three cell layers (Fig 3A⇑ and Fig 5A⇓). Immunofluorescence experiments carried out at the same stage demonstrated that the Cx40 protein was associated with the myocytes (Fig 6A⇓ through 6E). Cx40 was never detected in the endocardium, either at 9.5 dpc (compare panels E and F in Fig 6⇓) or at later developmental stages or in the adult heart. This held true for both atrial and ventricular endocardium. Between 9.5 dpc and 12.5 dpc, the atrial segmentation develops, and by the end of the 12.5 dpc stage, the atrium is completely partitioned off by the septum primum, leaving the left and right atria separated from each other, except for the interatrial foramen secundum. From 10.5 dpc onward, Cx40 was much more highly expressed in the atrial myocytes than at the previous stages (Fig 5C⇓, 5D⇓, and 5E⇓), and this expression level was seen to be maintained at the later developmental stages.13 No expression of the Cx40 gene was detected in the atrioventricular canal (Table 2⇓).

Whole-mount in situ hybridization. A and B, Transverse sections in a 9.5-dpc embryo hybridized with anti-sense Cx40 riboprobe. In panel A, f, ra, and la indicate the foregut and the right and left regions of the primitive atrium, respectively. Note that the Cx40 gene transcript is detected in the thin atrial wall (panel A). Panel B shows a section in the common ventricular chamber where a few myocytes in the primordial trabecular network exhibit hybridization signal. Bars=50 and 25 μm in panels A and B, respectively. C and D, Embryos (10.5 dpc) hybridized with anti-sense Cx40 riboprobe. Panel C shows a heart dissected from an embryo after hybridization. Note the strong signals in the right (ra) and left (la) atrial regions and the left ventricle (lv). The right ventricle (rv) is free of labeling. Panel D depicts a longitudinal section through a 10.5-dpc heart showing hybridization signals in the wall of the primitive atrium (ra and la) (filled with red blood cells) and the lv. No labeling was seen in serial sections of the rv. Arrow indicates the interventricular constriction. E and F, The tissues bordered with the two black boxes in panel D are shown at higher magnification in panel E (right part of the primitive atrium) and panel F (left ventricle). Note in panel F that activation of the Cx40 gene is at the highest in the outermost myocyte layers of ventricular tissue. Small arrows indicate endothelial cells. Bars=25 μm in panels E and F. G through I, Embryo (10.5 dpc) hybridized with anti-sense Cx37 riboprobe. Panel G shows a longitudinal section in the heart. The tissues bordered with the two black boxes in panel G are shown at higher magnification in panel H (ra) and panel I (lv). Note in panels H and I hybridization signals in the endothelial cells (arrows) lining the atrial wall and the ventricular trabecular network, respectively. Bars=12.5 and 25 μm in panels H and I, respectively. J through L, Embryos (10.5 dpc) hybridized with anti-sense Cx43 riboprobe. Panel J shows a longitudinal section in the heart. The tissues bordered with the two boxes in panel J are shown at higher magnification in panel K (ra) and L (lv). Note that the atrial tissue is free of labeling (panels J and K) while transcript is detectable in the ventricular wall (panel L). Bars=25 μm in panels K and L.

Expression of Cx37, Cx40, and Cx43 in the atrial tissue of mouse embryos. A through F, Frozen section in a 9.5-dpc embryo heart. Panels A and C are phase-contrast micrographs. Panel C is an enlargement of the rectangle shown in panel A. The section was treated with an anti-desmin mouse monoclonal antibody and anti-Cx40 rabbit polyclonal antibodies, bleached, and then treated again with an anti-PECAM rat monoclonal antibody (see “Materials and Methods”). The distribution of myocytes, identified by their immunoreactivity to anti-desmin antibody, is shown in the primitive atrium in panels B and D and in the common ventricular chamber in panel B. Cx40 is expressed in the myocytes (compare panel D, desmin, and panel E, Cx40), but it is not detected in the endothelial cells of the endocardium identified by their immunoreactivity to anti-PECAM antibody (compare panel E, Cx40, and panel F, PECAM). a indicates primitive atrium; en, endocardium; f, foregut; my, myocardium; and v, common ventricular chamber. Bar in A=100 μm (representative of panels A and B); bar in C=40 μm (representative of panels C through F). G through I, Frozen section in a 13.5-dpc embryo heart. Panel G is a phase-contrast micrograph. Panel H shows immunoreactivity of endocardial endothelial cells (identified with anti-PECAM antibodies, not shown) to anti-Cx37 antibodies in the atrium. Asterisk (in panels G, H, and I) indicates the epicardial face of the atrial tissue. Note the absence of immunoreactivity of this face devoid of endothelial cells. Panel I shows the expression of Cx43 in the epicardial and subepicardial layers and the trabecular network. Bar in G=40 μm (representative of panels G through I).

Spatiotemporal Distribution of the Three Proteins, Cx37, Cx40, and Cx43, at Different Stages of Mouse Cardiogenesis

The Cx37 gene transcript was detected in the primitive atrium at 9.5 dpc (Fig 3B⇑). At all stages investigated, hybridization and immunofluorescence signals specific for Cx37 gene products were seen in the atrial endocardium, associated with the endothelial cells (Fig 5G⇑ and 5H⇑ and Fig 6H⇑). In the endocardium lining the atrioventricular canal, Cx37 was detectable in the endothelial cells at the 10.5-dpc stage (Table 2⇑).

Expression of the Cx43 gene in the mouse embryo, and in particular in the heart, has previously been the subject of several detailed investigations.1119505159 Our results are in complete agreement with these investigations, and only the more pertinent points will be mentioned as a basis for comparison with Cx40 expression. No hybridization or immunoreactivity signal was observed in the atrium of 9.5- to 11.5-dpc embryos treated with the Cx43 riboprobe (Fig 3C⇑ and Fig 5J⇑ and 5K⇑) or anti-Cx43 antibodies. Cx43 gene expression in the atrium was detected as a weak hybridization signal from the 12.5-dpc stage only (not shown, but see References 50 and 515051 ). At the later developmental stages, Cx43 was shown to be expressed at a much higher level (Fig 6I). Cx43 gene expression was never detected in the atrioventricular canal, whatever the stage investigated (Table 2⇑).

Activation of the Cx40 Gene Is Delayed in the Myocytes of the Future Right Ventricle

At 9.5 dpc, the common ventricular chamber (ie, the primitive ventricle), formerly situated cephalically to the primitive atrium, is brought into its characteristic adult position, and it is demarcated from the bulbus cordis3560 by the interventricular constriction. The common ventricular chamber and the bulbus cordis will participate later in the formation of the left and right ventricles, respectively.35 At 9.5 dpc, the Cx43 gene transcript was undetectable or barely detectable in the primitive ventricle (Fig 3C⇑). However, evidence for activation of the Cx43 gene was provided by the immunoreactivity of ventricular outermost myocytes to anti-Cx43 antibodies (Fig 7⇓). Cx43 expression was limited to these cells; myocytes that formed the primordial trabecular network remained free of labeling (Fig 7⇓). Hybridization or immunofluorescence signals reflecting a very low activation of the Cx40 gene, dispersed in the primordial trabecular network of the ventricular chamber, were detected as early as 9.5 dpc (Fig 3A⇑ and Fig 5B⇑). The expression level of Cx37 gene transcript and protein in the endocardium of the ventricular regions was similar to that seen in the atrium (Fig 3B⇑ and Table 2⇑).

Expression of Cx43 in the ventricular tissue of 9.5-dpc mouse embryo. A, Phase-contrast micrograph of a frozen section through the common ventricular chamber. B, Immunoreactivity of the outermost cell layers to anti-Cx43 antibodies. No fluorescence signal was detected in the trabecular network (tra). Bar=15 μm.

From 10.5 dpc, the ventricular free wall thickens, the trabecular network develops, and the anlage of the septum forms at the level of the interventricular constriction. A striking feature at this stage was the strong activation of the Cx40 gene in the left ventricle, which was expressed by an accumulation of transcript in this region (Fig 5C⇑, 5D⇑, and 5F⇑). By contrast, no hybridization signal was detected in the right ventricle (Fig 5C⇑ and 5D⇑). As expected, immunofluorescence experiments revealed a prominent expression of Cx40 in the myocytes of the trabecular network and the compact free wall of the left ventricle (Fig 8A⇓ and 8B⇓), but no immunoreactivity was detected in the right ventricle at 10.5 dpc (Table 2⇑). At this stage, Cx43 gene expression, which was limited to the thin and compact ventricular free wall at 9.5 dpc, was seen to have spread, although very irregularly, to the myocytes of the trabecular network of both ventricles (Fig 5L⇑ and Fig 8A⇓ and 8C⇓). In the ventricular regions, where both Cx40 and Cx43 were expressed, they were colocalized in most of the gap junctions, as seen by conventional fluorescence microscopy (Fig 8B⇓ and 8C⇓) and confirmed by confocal microscopy analyses (not shown). In the endocardium, signals of hybridization and immunoreactivity of Cx37 gene products were of the same intensity as at the previous stage (9.5 dpc) (Fig 5G⇑ and 5I⇑ and Fig 8E⇓ and 8F⇓). Cx37 was detected in the endocardium from the subsequent developmental stages until adult stage.

Expression of Cx37, Cx40, and Cx43 in the ventricular tissue of 10.5-dpc mouse embryos in double-labeling immunofluorescence experiments. A through C, Frozen section through the trabecular network of the left ventricle. Panel A is a phase-contrast micrograph. Panels B and C show immunoreactivity of myocytes to anti-Cx40 and anti-Cx43 antibodies, respectively. Small arrows in panels B and C indicate gap junctions containing both Cx40 and Cx43. Arrowheads in the same panels show gap junctions labeled only with either one or the other of the antibodies. Bar in panel A=25 μm (representative of panels A through C). D through F, Frozen section through the trabecular network of the right ventricle. Panel D is a phase-contrast micrograph. Panels E and F show immunoreactivity to anti-CX37 and anti-PECAM antibodies, respectively. The dotted labeling of Cx37 is colocalized with PECAM expression (small arrows). Bar in panel D=25 μm (representative of panels D through F).

Cx40 was detected in the right ventricle from the 11.5-dpc stage. At this stage, and at the later stage of 12.5 dpc, Cx40 was seen colocalized with Cx43 in numerous gap junctions of the trabecular network and the free ventricular wall (Fig 9A⇓ through 9C). From 12.5 dpc, a gradient of expression of Cx40 appeared. Cx40 became barely detectable in the compact subepicardial layers, whereas its expression was maintained, if not increased, in the trabecular network, as illustrated in Fig 10A⇓ and 10B⇓ for the 14.5-dpc stage. A similar gradient was also observed for Cx43 (not shown, but see Reference 1919 ).

Expression of Cx40 and Cx43 in the right ventricle of 12.5-dpc mouse embryos. The section was treated with anti-Cx40 and anti-Cx43 antibodies, and then immunofluorescence signals were analyzed by confocal microscopy. A and B, Distribution of Cx40 (red) and Cx43 (green), respectively, in the gap junctions of the trabecular network. C, Merged picture of signals recorded in panels A and B. Yellow color indicates a colocalization of both Cxs. Note that in some junctions only Cx40 or Cx43 is detected (small arrows). Bar in panel A=25 μm (representative of panels A through C).

Expression of Cx37, Cx40, and Cx43 in the ventricular tissue of 14.5-dpc mouse embryos. A through C, Frozen sections through the left ventricle. Panel A is a phase-contrast micrograph of a section treated with both anti-Cx40 antibodies (panel B) and anti-desmin antibodies (not shown). Panel B shows immunofluorescence signals due to anti-CX40 antibodies. Panel C shows immunofluorescence signals due to anti-Cx37 antibodies in a section adjacent to the one shown in panel A and treated with both anti-Cx37 antibodies (panel C) and anti-PECAM antibodies (not shown). Vertical small arrows in panels A through C indicate the epicardium. Note in panel B the gradient of Cx40 expression, which decreases from the subepicardial layers (sep) to the trabeculae (tra). In the subepicardial layers, cords of anti-Cx40 antibodies immunoreactive sites (longitudinal arrows in panel B), parallel to the epicardium, suggest the presence of coronary vessels. This was confirmed by the arrangement of anti-Cx37 (panel C) and anti-PECAM (not shown) antibodies immunoreactive sites detected in the subepicardial layers of an adjacent section (panel C). Thus, panel C shows the distribution of Cx37 associated with endothelial cells of a coronary vessel. Unexpectedly, in this experiment, Cx37 was barely detectable in the endothelial cells of endocardium, which lies in the trabeculae present in the lower quarter of the section shown in panel C. Bar in panel A=40 μm (representative of panels A through C). D through F, Frozen longitudinal sections in the interventricular septum (ivs) from a double-labeling immunofluorescence experiment. Only the anterior region of the ivs is shown. Asterisk indicates the interventricular foramen normally closed at this stage by a membranous structure, which disappeared during the preparation of the specimen. The section shown in the phase-contrast micrograph of panel D was treated with both anti-CX43 and anti-Cx40 antibodies. Panel E shows immunoreactivity of peripheral myocytes to anti-CX40 antibodies. Arrow shows the localization of the primitive His bundle, at the top of the ivs. Panel F shows immunoreactivity to anti-Cx43 antibodies. Only the inner myocytes, intrinsic to the ivs, express Cx43. Comparison of the panels E and F shows clearly the complementary expressions of Cx40 and Cx43. Bar in panel D=40 μm (representative of panels D through F).

Between 10.5 and 14.5 dpc, the interventricular septum develops, and by 14.5 dpc, the septation of the ventricles is almost complete. The heart has achieved the definitive arrangement that will persist until birth. Cx40 expression was never observed in the myocytes of the interventricular septum (Fig 10D⇑ through 10F and Table 2⇑). In contrast, from 13.5 dpc, the peripheral myocytes located at the very top of the septum and lying on its flanks, ie, the myocytes forming the primitive conduction system, were seen to highly express Cx40 (Fig 10D⇑ through 10F). Remarkably, Cx43 was neither detected in the myocytes of the primitive His bundle nor in those of the anterior regions of the primitive bundle branches (Fig 10E⇑ and 10F⇑). This differential expression of Cx40 and Cx43 in the anterior region of the conduction system persisted at the subsequent developmental stages and was also observed in the adult heart (not shown). The latter feature has previously been described in adult and embryonic rat hearts.1020212232 Beyond 14.5 dpc, the progressive spatiotemporal restriction of the Cx40 expression and, in parallel, the upregulation of Cx43 have been described by Delorme et al13 and Fromaget et al,19 respectively. Finally, it should be pointed out that neither the Cx40 gene products nor those of the Cx43 gene were ever observed in the outflow tract myocardium (as recently defined),60 whatever the developmental stages, in agreement with the results reported for the rat heart2022 (Table 2⇑).

During heart maturation, the coronary vessels differentiate in the compact subepicardial layers from precursor cells migrating from the liver region.61 In the 10.5-dpc mouse heart, Virágh and Challice62 have described the appearance of vascular buds, which were the earliest signs of coronary vessel formation. From 13.5 to 14.5 dpc, when the gradient of Cx40 expression has been established, discrete, cordlike anti-Cx40 immunoreactive sites were observed in the subepicardial layers (Fig 10A⇑ and 10B⇑). The topology of these sites, parallel to the epicardium, suggested the presence of vascular channels, precursors of coronary vessels formed by endothelial cells. This was confirmed by the immunoreactivity of channel-forming cells to anti-Cx37 (Fig 10C⇑) and anti-PECAM antibodies (not shown).

Discussion

The present work combined RT-PCR, in situ hybridization, and immunofluorescence techniques to investigate the expression and regulation of three Cxs (Cx37, Cx40, and Cx43) during the early stages of mouse heart maturation. We have demonstrated that Cx37 was expressed in endothelial cells of both blood vessels and endocardium and that expression of the Cx40 gene in myocytes depends on a unique activation program. Three points will be discussed below regarding (1) the Cxs in the endothelial cells, (2) the regionalization of Cx40 gene expression in heart, and (3) the redundancy and complementarity of Cx40 and Cx43 gene expression in myocytes of different cardiac regions.

Cxs in the Endothelial Cells

Expression of Cx37 in vascular endothelial cells is consistent with the presence of gap junctions between these cells,6364 and this finding is in agreement with various results reporting the detection of Cx37 mRNA and/or Cx37 protein in cultured or in situ vascular endothelial cells.656667 In contrast, the expression of Cx37 in the endocardium has not yet been reported, and it is so far the only Cx detected in this thin tissue layer. Thus, Cx37 would appear to be involved either alone or with other as-yet-unidentified Cxs in the formation of gap junctions seen between endocardial endothelial cells.6869 Besides Cx37, a second Cx, Cx40, was found to be expressed in vascular endothelial cells of the mouse embryo in general and of myocardium in particular (coronary vessels). These results are in agreement with previously published reports that described in various species Cx40 expression in the endothelial cells of the vascular tree.313259677071 It is not yet known, however, whether Cx37 and Cx40, which are compatible Cxs,333 are expressed in the same endothelial cells and, if they are, whether they are colocalized in the same gap junctions. Cx40 was found not to be expressed in the atrial and ventricular endocardium. This Cx could be considered as a marker of the vascular endothelial cells. Consequently, a detailed investigation (which would go well beyond the scope of the present study) of the expression of Cx40 and Cx37 in the endothelial cells of the outflow tract area and the heart venous pole would perhaps make it possible to answer the question that is posed by embryologists concerning the boundary between vascular endothelium and endocardial endothelium.

Our results demonstrate the early expression of Cxs in endothelial cells of mouse embryos, and of the three Cx genes investigated, the Cx40 gene was shown to be the first gene activated in the vascular system: its transcript was detectable, weakly, by in situ hybridization as early as the 8.5-dpc stage (data not shown). From 9.5 dpc onward, both Cx40 and Cx37 were detected in endothelial cells of developing and remodeling large vessels, and when the myocardial vascularization was initiated, they were also both expressed in the coronary vessels. Thus, early in development, Cx37 and Cx40 were demonstrated to be closely associated with the vascularization process. Both Cxs were also seen in the vascular endothelial cells in the adult, but they were never detected in heart capillaries, which perhaps indicated a very low Cx expression level in these structures. No hybridization or immunoreactivity signal suggesting the expression of Cx43 in vascular endothelial cells was ever detected in mouse embryos or in adult hearts. Cx43 expression has, however, been described in endothelial cells of certain regions of the vascular tree in other species, such as rats72 and humans.67

The physiological role of gap junctions in the coordinated contraction of uterine smooth muscle cells73 and cardiac myocytes14 is well documented. Their role is much less clear in vascular endothelium. Vascular endothelial cells communicate via electrical and dye coupling,65666774 and it has recently been demonstrated that their gap junctions are permeable to inositol 1,4,5-trisphosphate.66 Junctional communication between these cells is thought to be involved in their migration capacity during wound repair, but our results suggest they also have a potential role in vasculogenesis and angiogenesis. Another aspect that has to be considered regarding the vessels is the junctional communication between vascular endothelial cells and subjacent smooth muscle cells of the tunica media. This heterotypic communication, demonstrated by several authors, would appear to be implicated in the coordination of vasomotor responses (for reviews, see References 75 and 767576 ). To our knowledge, the physiological role of gap junctions in endocardial endothelial cells has not yet been addressed, but if it is confirmed that gap junctions between these cells are constituted only of Cx37, then endocardium endothelial cells would be a suitable model for the investigation of Cx37 endogenous channels.

Regionalization of Cx40 Gene Expression in Heart

During the developmental period covered by the present investigation (8.5 to 14.5 dpc), Cx40 gene expression in mouse heart myocytes is subordinated to strong spatiotemporal regulation. The Cx40 gene was shown to be transcribed at 8.5 dpc, but its mRNA was present in very low amounts, and it was detectable only by RT-PCR. At 9.5 dpc, the gene was found to be activated: its transcript and the protein were both detected by in situ hybridization and immunofluorescence in the primitive atrium and, to a lesser extent, in the common ventricular chamber. Activation of the Cx40 gene in the embryonic right ventricle was seen from 11.5 dpc only. Beyond this stage and until 14 to 14.5 dpc, atria and ventricles equally expressed Cx40, except in the interventricular septum, where Cx40 gene expression was never detected (Table 2⇑). Thus, at the early stages of mouse heart maturation, Cx40 gene activation proceeds according to a caudorostral gradient involving first the primitive atrium and the left ventricular region and then the right ventricular region, while avoiding, however, the atrioventricular canal and the interventricular septum. During this period, there is clearly a temporary regionalization of the Cx40 gene expression. The regionalization of this Cx is in fact superimposed on a morphological and functional regionalization of the myocardium, which (in chickens but probably also in rodents, although this has yet to be formally demonstrated) sees the alternating of segments with slow- and fast-conducting properties.77 The slow-conducting inflow tract, the fast-conducting atrium, the slow-conducting atrioventricular canal, the fast-conducting ventricle, and, finally, the slow-conducting outflow tract follow each other, in the heart, in the caudorostral direction.77 During the heart morphogenesis, as shown in Table 2⇑ for stages 9.5 to 14.5 dpc, expression of Cx40, and also of Cx43, is associated with the fast-conducting regions. From 14 to 14.5 dpc, the Cx40 gene expression is modified in space and time, as previously described,13 leading to a further regionalization of Cx40 in the adult heart. This regionalization, which has been demonstrated in the adult myocardium of other mammal species, again associates the expression of Cx40 with domains of fast conduction.21

Redundancy and Complementarity of Cx Gene Expression in Heart

Homozygous Cx43−/− mouse embryos die shortly after birth because of their inability to establish autonomous pulmonary circulation. Death is the consequence of an obstruction of the outflow tract due to a proliferation of myocytes, producing interconnected septa and blind-ended cavities, in the right ventricular chamber.24 Before birth, the heart contracts rhythmically,78 which implies that one or several Cxs substitute for Cx43 in its current-carrying capacity. Among the known cardiac Cxs, two are candidates for this substitution role: Cx40 and Cx45. The distribution of Cx40 in embryonic, newborn, and adult mouse heart is known (the present study and Delorme et al13 ), and this Cx, highly expressed in embryonic heart, could replace Cx43 functionally. However, whatever the developmental stage considered, the expressions of Cx40 and Cx43 are never overlapping in all cardiac tissues. Immunofluorescence investigations have shown, for example, that in the late fetal stages and at birth, Cx40 is not expressed in the interventricular septum and the ventricular free walls. In the light of these results, it is difficult to explain the propagation of electrical activity in the ventricular walls of the homozygous neonates for the Cx43 null mutation,79 especially since Cx40 regulation in heart of mutant embryos is identical to that of wild-type embryos (J. Ya, A.F.M. Moorman, W.H. Lamers, unpublished data, 1997). The simplest hypothesis to explain the persistence of the conduction phenomenon is to postulate that a Cx other than Cx40 is involved in the electrical coupling between myocytes, at least in heart regions lacking Cx40. Cx45, whose expression is not upregulated in the myocardium of Cx43 knockout mouse embryos (J. Ya, A.F.M. Moorman, W.H. Lamers, unpublished data, 1997), could be this Cx, and consequently, its distribution in the developing heart deserves particular consideration. Finally, the possibility that another Cx, which is not normally expressed in the myocardium, could be upregulated in the heart of Cx43−/− mice cannot be excluded.

Looping of the heart to the right is the first indication of left-right asymmetry in the embryo. Several signaling molecules and extracellular matrix molecules that may play a role in cardiac looping have been identified recently (for reviews, see References 80 and 818081 ). Within this context, it is interesting to observe that at the early stages of heart maturation, ie, between 9.5 and 11.5 dpc, Cx40 is expressed only in one of the two compartments (the common ventricular chamber) that will participate in the formation of the ventricles. In contrast, at the same stages, Cx43 is expressed in both ventricles. Whether this asymmetrical expression of Cx40 may play a role in the morphogenesis of the heart is not known.

Selected Abbreviations and Acronyms

Cx

=

connexin

dpc

=

day(s) post coitum

DTAF

=

dichlorotriazinyl-aminofluorescein

FITC

=

fluorescein isothiocyanate

PCR

=

polymerase chain reaction

PECAM

=

platelet endothelial cell adhesion molecule

RT

=

reverse transcriptase

Tm

=

melting temperature

TRITC

=

tetraethylrhodamine isothiocyanate

Acknowledgments

These investigations were supported by grants from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche (grant ACC SV No. 95-09003, Dr Gros), the Association Française contre les Myopathies (Dr Gros), the European Economic Community (grant BMH4-CT 96-1427, Drs Gros and Willecke), and the Deutsche Forschungsgemeinschaft SFB 284 C1 (Dr Willecke). We thank C. Moretti, P. Weber, G. Turini, and N. Barrois for their valuable assistance in image analysis and photography. We thank also Drs W. Lamers, H.J. Jongsma, and P. Durbec for the critical reading of the manuscript and their suggestions.